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Abstract

Etimicin (ETM) can accumulate in kidneys and cause tubular epithelial cell cytotoxicity. This article aims to study ETM elimination in kidneys and its nephrotoxicity, apoptosis, and histopathological insults of renal tubular epithelial cells, after repeated administration. A total of 36 rats were randomly divided into ETM-treated group and vehicle control group. Rats in ETM-treated group were treated intraperitoneally (i.p.) with 100 mg/kg/day ETM and rats in control group received physiological saline (i.p.) for 5 consecutive days. Determination of ETM concentrations accumulated in rat kidneys was carried out by high-performance liquid chromatography on the basis of derivatization with o-phthalaldehyde and by ultraviolet detector. Apoptotic renal tubular epithelial cells were identified by a terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assay. Histopathological insults in kidneys were evaluated by hematoxylin and eosin staining. On day 1 after cessation of ETM administration, the accumulation concentration was 347.50 ± 193.30 μg/g tissue; on day 15, ETM concentration became 16.71 ± 9.99 μg/g tissue. Elimination half-life of ETM in rat kidney was about 3.05 days. Apoptotic renal tubular epithelial cells induced by etimicin was recovered gradually from 1544 ± 138 n/mm² on day 1 to 716 ± 208 n/mm² on day 15. Histopathological damage was also gradually recovered from vacuolation of tubular epithelial cells as well as renal tubular edema on days 1, 3, and 7 to nearly normal on day 15. From these results, we concluded that renal tubular epithelial cell cytotoxicity induced by ETM can gradually restore with its decreasing concentration in rat kidneys.

Keywords

Etimicin nephrotoxicity renal tubular epithelial cell apoptosis

Introduction

Etimicin (ETM), which is originally named 89-07, is a new semisynthetic aminoglycoside antibiotic obtained by chemical modification of gentamycin C_1a ¹ and was found better active against both gram-positive and gram-negative bacteria, including the bacterial strains resistant to other aminoglycoside members such as gentamicin and amikacin.² Besides, ETM still has nephrotoxicity, especially to elderly or patients with renal dysfunction at a lower rate,³ though nephrotoxicity of ETM is lower than that of other aminoglycoside antibiotics by routine dosage.⁴

Aminoglycoside antibiotics nephrotoxicity is typically characterized by tubular damage arising from tubular epithelial cell cytotoxicity.⁵ Apoptosis as well as necrosis of tubular epithelial cells appears in experimental animals treated by gentamicin in vivo and also in cultured cells.^6
–8 For other toxins, such as chemotherapeutic agents and hydrogen peroxide,^9,10 a relationship exists between toxin concentration and death phenotype. Therefore, the objective of this study is to observe the correlation between ETM elimination in renal tissue and cytotoxicity, apoptosis, and pathological injuries of renal tubular epithelial cells, in order to provide reference proof for clinical treatment.

Materials and methods

Main drugs and reagents

Standard etimicin sulfate and etimicin sulfate injection were obtained from Changzhou Fangyuan Pharmaceutical Co. Ltd (Jiangsu, China). Boric acid was obtained from Chemical Plant (Beijing, China). Analytical-grade thioglycolic acid and o-phthalaldehyde were obtained from Sinopharm Chemical Reagent Co. Ltd (Beijing, China). Sodium heptanesulfonate of high-performance liquid chromatography (HPLC) grade was obtained from Dikma Technology Inc. (Lake forest, California, USA). In Situ Cell Death Detection Kit, Fluorescein was obtained from Roche (San Francisco, California, USA).

Animals

Healthy adult male Wistar albino rats of specific pathogen-free grade weighing 180 ± 10 g were purchased from the Laboratory Animal Center of the Academy of Military Medical Sciences (Certificate No. SCXK (Army) 2007-004). All the animals were housed in clean plastic cages maintained under standard conditions (12-h light and 12-h dark, 25 ± 5°C, and 35–60% humidity) with free access to tap water and standard rat diet throughout the experiment. An acclimatization period of 2 days was allowed to the rats before any experiment. The Institutional Animal Care and Use Committee of General Hospital of Air Force approved the use of animals, and the experimental protocol of animal was designed in accordance with the Guide for the Care and Use of Laboratory Animals of Research Council.

Animal experimental design

The rats (n = 36) were divided into ETM-treated group including 30 rats injected intraperitoneally (i.p.)¹¹ with ETM of 100 mg/kg body weight/day for 5 consecutive days and vehicle control group including 6 rats injected i.p. with equal volume of physiological saline. A total of six rats in ETM-treated group were respectively killed on days 1, 3, 7, 10, and 15 after cessation of ETM repetitive administration and their kidneys were gathered. Rats in control group were killed on day 1 after the last dosing.

Kidney handling

The rats were deeply anesthetized with intraperitoneal injection of ketamine (120 mg/kg body weight). Kidneys were harvested after each rat was killed. The left kidneys were stored in physiological saline solution to determine the contents of ETM accumulated in kidneys. The right kidneys were separated into two nearly equal parts along longitudinal section. One part wrapped with germ-free gauze moistened by physiologic saline and frozen at −20°C was used for detection of apoptosis. The other part fixed in 4% neutral formalin solution was used for histopathological investigations.

Determination of ETM concentration in kidneys

ETM concentrations accumulated in kidneys were analyzed by high-performance liquid chromatography (HPLC) on the basis of derivatization with o-phthalaldehyde and by ultraviolet detector according to the method reported by Guo et al.¹² Briefly, the kidneys were weighed and homogenized according to 1: 8 (g:mL) ratio of normal saline. Renal tissue homogenate (1.0 mL) was centrifuged for 10 min at 6,640g, followed by 400 μL of supernatant transferred to a microcentrifuge tube, into which 700 μL of isopropanol was added. The microcentrifuge tube was oscillated for 30 s and centrifuged at 6,640g for 10 min, whose upper supernatant was kept for subsequent analysis. The column type used in HPLC analysis was Platisil C₁₈ (150 × 4.6 mm², 5 μm; Dikma Technologies) with Easy Guard C₁₈ precolumn, the column temperature was set at 35°C, flow rate was 1.0 mL/min, retention time was 9.1 min, and mobile phase was 0.02 mol/L sodium heptanesulfonate diluted by a mixture consisting of 20 volumes of water, 5 volumes of acetic acid, and 75 volumes of methanol.

Apoptosis assay

Apoptosis in renal tubular epithelial cells was identified by a terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling (TUNEL) assay in situ according to the manufacturer’s instructions.^13,14 Briefly, the refrigerant kidney specimens were cut into 5-μm sections and mounted onto the glass slides. The sections were treated with freshly prepared fixation solution for 20 min, washed with phosphate-buffered saline (PBS) for 30 min under 15–25°C, followed by incubating with permeabilization solution for 2 min on ice (2–8°C). The tissue slices were washed three times with PBS and incubated with TUNEL reaction mixture in a humidified atmosphere in the dark.

For each slice, five representative and nonoverlapping vision fields were chosen from center and four corners to be observed under a 400-fold fluorescence microscope. The green-colored granular cells were considered as TUNEL-positive cells and analyzed with CMIAS-II image analysis system in a blind fashion.¹⁵ Data are expressed as the number of apoptotic cells per quadrat millimeter (n/mm²).

Histopathological examinations

The renal tissues fixed in 4% neutral formalin solution were processed conventionally for light microscopic observation according to a standard pathological procedure.¹⁶ A semiquantitative evaluation of renal tissue pathological insults was performed by previously published criteria.¹⁷ A total of six indexes, namely tubular brush border loss, renal tubular edema, tubule epithelial cell vacuolar degeneration, renal tubular epithelial cell necrosis, renal tubular dilatation, and interstitial inflammation were used to evaluate nephrotoxicity. Score obtained was defined as follows: 0 = no pathological change and 1 = appearance of any one of above six indexes. Five representative and nonoverlapping vision fields were also chosen from center and four corners on slice to be used for evaluating scores according to the above six indexes by one pathologist in a blinded fashion. The total scores in each vision field were got by adding up score of above six indexes. The higher the total scores were, the severer the renal injury would be.

Statistical analysis

The quantitative data obtained were expressed as mean ± SD and analyzed by one-way analysis of variance using Statistical Package for Social Sciences software (version 17.0; SPSS Inc., Chicago, Illinois, USA). A difference was assumed to be statistically significant when p < 0.05 or 0.01.

Results

Elimination of ETM in rat kidneys

ETM concentrations in kidneys at various time points (Table 1) were analyzed by HPLC on the basis of derivatization with o-phthalaldehyde. The maximal accumulation concentration was reached on day 1 after cessation of ETM repetitive administration, which was 347.50 ± 193.30 μg/g tissue and thereafter began to decrease progressively. From day 7onward, ETM concentration accumulated in renal tissues was reduced significantly as compared to that on day 1 (p < 0.01) or day 3 (p < 0.01).

Table 1.

The concentration elimination of ETM accumulated in rat kidneys.^a

Day^b	Renal weight (g)	ETM contents (μg)	ETM concentrations (μg/g tissue)
1	1.06 ± 0.04	370.20 ± 208.60	347.50 ± 193.30^c
3	1.11 ± 0.08	359.80 ± 135.50	328.10 ± 125.40^c
7	1.25 ± 0.02	138.10 ± 64.64	110.20 ± 51.14
10	1.32 ± 0.04	73.02 ± 20.47	55.04 ± 14.84
15	1.11 ± 0.13	17.90 ± 9.46	16.71 ± 9.99
Control	1.25 ± 0.05	0	0

ETM: etimicin.

^aValues were expressed as mean ± SD for 6 animals in each group.

^bTime after cessation of ETM administration.

^c p < 0.01: statistically significant difference from days 7, 10, and 15 group.

Residual method was used to calculate elimination half-life (t _1/2) of ETM in rat kidney tissues. The logarithmic concentrations of ETM accumulated in rat kidney tissues at different time points were noted (Table 2). A straight line could be constructed by plotting logarithmic concentrations of ETM in rat kidney tissues (y) versus the time (x) (Figure 1). The straight line equation was y = −0.0987x + 2.723 and elimination rate constant (k) obtained was 0.227. According to the formula, t _1/2 = 0.693/k, t _1/2 we got was 3.05 days, that is, the t _1/2 of ETM in rat kidney tissues was about 3.05 days.

Figure 1.

Relationship between logarithmic concentrations of ETM in rat kidneys and the time after the cessation of ETM treatment. ETM: etimicin.

Table 2.

The logarithmic concentrations of ETM accumulated in rat kidneys at different time points (n = 6).

Time (days)^a	ETM concentrations (μg/g)	Logarithmic concentrations
1	347.50 ± 193.30	2.54
3	328.10 ± 125.40	2.52
7	110.20 ± 51.14	2.04
10	55.04 ± 14.84	1.74
15	16.71 ± 9.99	1.22

ETM: etimicin.

^aTime after cessation of ETM treatment.

Renal tubular epithelial cell apoptosis induced by ETM accumulated in kidney

As determined by TUNEL staining, the green-colored fluorescence was determined as positive staining of apoptotic cells. Figure 2 (upper) shows that renal tubular epithelial cells of rats treated with vehicle showed few apoptotic cells, but treatment with ETM for 5 consecutive days underwent notable cell apoptosis. Additionally, cell apoptosis on day 3 after ETM treatment was visually severe than that on other days. There were still a few more detectable staining cells on day 15 after ETM termination.

Figure 2.

The apoptotic images (upper image: TUNEL assay, ×400) and quantification (lower image) of rat tubular epithelial cells. Values were represented the mean ± SD for six animals. (A) Day 1 after cessation of ETM treatment, (B) day 3, (C) day 7, (D) day 10, (E) day 15, and (F) vehicle control. *p < 0.05: significantly different as compared to days 7 and 15 group. ETM: etimicin; TUNEL: terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling.

On days 1, 3, 7, 10, and 15 after cessation of ETM administration, apoptotic cells analyzed by MIAS software were 1544 ± 138, 1858 ± 701, 1596 ± 382, 855 ± 92, and 716 ± 208 n/mm², respectively. From day 10 onward, apoptotic renal tubular epithelial cells displayed a dramatic decrease compared with that on day 3 (p < 0.05), displaying a significantly descending tendency (Figure 2 lower).

Linear relationship between ETM elimination and apoptotic renal tubular epithelial cells at different time points

Linear relationship was constructed between apoptotic renal tubular epithelial cells and the concentrations of ETM in kidneys at different time points. Figure 3 shows a better linear relationship (r = 0.82, p = 0.059) between ETM concentration and apoptotic renal tubular epithelial cells.

Figure 3.

Relationship between ETM concentrations and apoptotic renal tubular epithelial cells. ETM: etimicin.

Histopathologic changes

The histopathological changes in kidneys were evaluated as described in the Materials and Methods section and the degrees of histopathological insults were summarized in Table 3. As shown in Figure 4, rats receiving saline injection revealed normal renal glomeruli and renal tubules under fluorescent microscope (Figure 4(F)). However, on day 1 after ETM treatment, pathological changes were vacuolar degeneration of tubular epithelial cells and renal tubular edema (Figure 4(A)). On day 3, vacuolar degeneration, renal tubular dilatation, and mild inflammatory cell infiltration were presented (Figure 4(B)). On day 7, renal tubular edema and inflammatory cell infiltration were found (Figure 4(C)). On day 10, kidney damages induced by ETM were weakened obviously as kidney morphology appeared almost normal except only renal tubular dilatation (Figure 4(D)). On day 15, kidney structures of rats (Figure 4(E)) were nearly similar to those in normal saline control group (Figure 4(F)).

Figure 4.

Histopathological observation of renal sections at different time points (H&E staining, ×400). (A) Day 1 after cessation of ETM treatment, (B) day 3, (C) day 7, (D) day 10, (E) day 15, and (F) vehicle control. e: renal tubular edema; i: interstitial inflammation;t: renal tubular dilatation; v: tubule epithelial cell vacuolar degeneration; ETM: etimicin; H&E: hematoxylin and eosin.

Table 3.

Semiquantitative analysis of renal tissue insult of rats in each group.^a

Days^b	Score						Total score
Days^b	(a)	(b)	(c)	(d)	(e)	(f)	Total score
1	0.0 ± 0.0	0.4 ± 0.5	0.3 ± 0.5	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.7 ± 0.2
3	0.0 ± 0.0	0.0 ± 0.0	0.4 ± 0.5	0.0 ± 0.0	0.5 ± 0.5	0.1 ± 0.3	1.1 ± 0.2^c
7	0.0 ± 0.0	0.4 ± 0.5	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.4 ± 0.5	0.8 ± 0.2
10	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.5 ± 0.5	0.0 ± 0.0	0.5 ± 0.2
15	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0
Control	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0

ETM: etimicin; a: tubular brush border loss; b: renal tubular edema; c: tubule epithelial cell vacuolar degeneration; d: renal tubular epithelial cell necrosis; e: renal tubular dilatation; f: interstitial inflammation.

^aValues were expressed as mean ± SD for six animals in each group.

^bTime after cessation of ETM treatment.

^c p < 0.01: statistically significant difference from days 10 and 15 group.

Semiquantitative analysis displayed that scores of renal tissue insult after ETM treatment on day 1 was 0.7 ± 0.2, on day 3 was 1.1 ± 0.2, on day 7 was 0.8 ± 0.2, on day 10 was 0.5 ± 0.2, and on day 15 was 0, indicating that pathological insults can restore to normal status (Table 3).

Discussion

ETM, one of aminoglycoside antibiotics, is commonly used in the treatment of life-threatening infections. However, this antibiotic is found to induce nephrotoxicity, especially renal tubular epithelial cell cytotoxicity, by its accumulation in kidneys after repeated administration, which seriously limits its optimal clinical usefulness.^18
–20 So it is essential to present the information that how long ETM content accumulated in kidney after repetitive administration will be completely eliminated.

ETM is commonly used intravenously with 300 mg/day once a day for approximately 5–7 days in clinical practice, by which we designed the present experiment. Data in the present study displayed that ETM contents accumulated in kidneys of rats injected i.p. for 5 days showed a continuous decrease from day 1 (347.50 ± 193.30 μg/g) to day 15 (16.71 ± 9.99 μg/g) after cessation of ETM treatment, which was similar to the results reported by Guo et al.,¹² who found that ETM concentration accumulated in renal tissues decreased gradually from day 1 (31.2 ± 9.2 μg/g) to day 10 (2.8 μg/g) after cessation of ETM injected intramuscularly by a single-dosage (2 mg/kg body weight). The cause of the phenomenon may be that the megalin localized on apical membrane, a giant glycoprotein (approximately 600 kDa) receptor with many ligands such as aminoglycosides, is involved in the transport of ligand, ETM, to the opposite plasma membrane and its release to the extracellular environment without degradation.²¹ Another cause may be that it is degraded by cytochrome P450 in kidney. Detailed causes remained to be explored further.

El Mouedden et al.²² reported that treatment with gentamicin (10 mg/kg body weight/day) for 10 days induced a marked apoptosis of renal tubular epithelial cells of Wistar rats. Liu et al.²³ reported that gentamicin (100 mg/kg body weight/day) injected i.p. for 5, 8, and 10 days, respectively, were all found to induce notable renal tubular epithelial cell apoptosis in rats. These results showed that renal tubular epithelial cell apoptosis may be an important mechanism of gentamicin nephrotoxicity. In our study, ETM also induced renal tubular epithelial cell apoptosis on various days after cessation of treatment at dose of 100 mg/kg body weight /day for 5 days, which indicated that apoptosis of renal tubular epithelial cells may be also an important nephrotoxic mechanism of ETM. Because ETM and gentamicin closely resemble each other in biochemical characteristics²⁴ and pharmacokinetics²⁵ besides chemical structure, we speculated that the apoptotic mechanisms induced by ETM was similar to those induced by gentamicin which in the cytosol acted on mitochondria in a direct and also in an indirect fashion and triggered the translocation of cytochrome c and other proapoptotic proteins such as apoptosis-inducing factor in mitochondria. Cytochrome c in the cytosol activates caspase 9 and, finally, the executor caspases 3 and 7, which results in cellular death by apoptosis.⁵

Results from our study showed a marked recovery trend of tubular epithelial cell apoptosis to normal after rats were injected i.p. with ETM of 100 mg/kg body weight/day for 5 consecutive days. This finding can be related to decreasing concentration of ETM in renal tissues because better linear correlation was revealed between apoptotic renal tubular epithelial cells induced by ETM and its elimination in kidneys (r = 0.82, p = 0.059).

In this study, histopathological insults were mainly vacuolar degeneration of renal tubular epithelial cell as well as renal tubular edema, which recovered to normal on day 15 after cessation of administration. These results were similar to the findings reported by Li et al.²⁰ but were significantly different from the results reported by Payasi et al.,²⁶ who found that there were no obviously histopathological toxicity changes observed in mice with the treatment of ETM up to the dose of 200 mg/kg body weight/day for 28 days. The cause of the difference may be related to different animal species that had different reaction to the drug. From these results, pathological insults were not believed to be the main phenotype of renal tubular epithelial cell death induced by ETM.

Conclusion

The results in this study provide the evidence that renal tubular epithelial cell cytotoxicity induced by ETM can gradually restore with its decreasing concentration in renal tissues.

Footnotes

Conflict of interest

The authors declared no conflicts of interest.

Funding

This work was supported by the Natural Science Foundation of PLA, China (grant no. 06MA024).

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Elimination of etimicin in rat kidneys and alterations of its cytotoxicity to tubular epithelial cells

Abstract

Keywords

Introduction

Materials and methods

Main drugs and reagents

Animals

Animal experimental design

Kidney handling

Determination of ETM concentration in kidneys

Apoptosis assay

Histopathological examinations

Statistical analysis

Results

Elimination of ETM in rat kidneys

Renal tubular epithelial cell apoptosis induced by ETM accumulated in kidney

Linear relationship between ETM elimination and apoptotic renal tubular epithelial cells at different time points

Histopathologic changes

Discussion

Conclusion

Footnotes

Conflict of interest

Funding

References